In contact hypersensitivity (CHS), multiple cells, inflammatory mediators and cytokines are known to be involved in the regulation of the immune response. Previously, we revealed the reactive oxygen species generation by 2, 4, 6-trinitrobenzene sulphonic acid (TNBS) in vivo, followed by heat shock protein 70 (Hsp70) carbonylation and the exogenous antioxidant role of cell-permeable Hsp70. Here, we demonstrate the role of Hsp70 using cell-permeable Hsp70 in the mouse CHS model. Pretreatment of cell-permeable Hsp70: (i) suppressed ear swelling; (ii) down-regulated phosphorylated p38, but up-regulated phosphorylated extracellular signal-regulated kinase; (iii) increased population of CD4(+) CD25(+) Foxp3(+) T cells; (iv) decreased secretion of tumor necrosis factor-α (TNF-α), IL-12, interferon-γ and IL-2 and (v) but up-regulated IL-4 and transforming growth factor beta (TGF-β) in the lymph nodes. In conclusion, cell-permeable Hsp70 attenuates CHS through modulation of MAPK pathway and regulation of Th1, Th2 and regulatory T cells.